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WO2008106149A1 - Procédés de prédiction du début de la ménarche - Google Patents

Procédés de prédiction du début de la ménarche Download PDF

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Publication number
WO2008106149A1
WO2008106149A1 PCT/US2008/002566 US2008002566W WO2008106149A1 WO 2008106149 A1 WO2008106149 A1 WO 2008106149A1 US 2008002566 W US2008002566 W US 2008002566W WO 2008106149 A1 WO2008106149 A1 WO 2008106149A1
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WIPO (PCT)
Prior art keywords
value
time interval
hormone
days
menarche
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PCT/US2008/002566
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English (en)
Inventor
Marc Laufer
Henry Feldman
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Boston Childrens Hospital
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Boston Childrens Hospital
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Priority to US12/528,814 priority Critical patent/US20100105071A1/en
Publication of WO2008106149A1 publication Critical patent/WO2008106149A1/fr
Anticipated expiration legal-status Critical
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • G01N33/743—Steroid hormones
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/36—Gynecology or obstetrics
    • G01N2800/361—Menstrual abnormalities or abnormal uterine bleeding, e.g. dysmenorrhea
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/60—Complex ways of combining multiple protein biomarkers for diagnosis

Definitions

  • Embodiments of the invention described herein relate to methods for predicting the onset of menarche through the measurement of salivary hormone levels (e.g. 17- ⁇ estradiol, testosterone, progesterone, and/or 17 hydroxy-progesterone (17-OHP)).
  • salivary hormone levels e.g. 17- ⁇ estradiol, testosterone, progesterone, and/or 17 hydroxy-progesterone (17-OHP)
  • the methods described herein enable a reliable determination that menarche will, or will not occur, within an identified time interval, e.g. 1 to 30 days, 1 to 45 days, 1 to 60 days and 1 to 90 days. Diagnostic kits and products of manufacture comprising these kits are also provided. BACKGROUND OF THE INVENTION
  • Prediction of menarche is commonly based on clinical conjecture, using an imprecise combination of Tanner staging, weight, and body-mass index (BMI).
  • BMI body-mass index
  • the ability to predict the onset of menses is important both from a medical standpoint e.g, to avoid the occurrence of bleeding during throbocytopenia of cancer chemotherapy; and from a psycho-social standpoint e.g., to decrease anxiety over timing of the occurrence of menarche such as when attending summer camp, or during a sports competition.
  • a medical standpoint e.g. to avoid the occurrence of bleeding during throbocytopenia of cancer chemotherapy
  • a psycho-social standpoint e.g., to decrease anxiety over timing of the occurrence of menarche such as when attending summer camp, or during a sports competition.
  • the present invention provides methods for predicting the onset of menarche in girls and young women within short time intervals including, but not limited to, 1 to 30 days, 1 to 45 days, 1 to 60 days and 1 to 90 days.
  • the diagnostic methods involve measuring the level of at least one hormone (e.g. 17- ⁇ estradiol, testosterone, progesterone, and/or 17 hydroxy- progesterone (17-OHP)) in saliva from a female.
  • the measured hormone levels are compared to a predetermined threshold level, H*, for at least one hormone, where a measured hormone level above the threshold hormone level indicates that menarche will occur within the specified interval.
  • a measured hormone level below a predetermined threshold hormone level indicates that menarche will not occur in the specified time interval.
  • the measured level is applied to a multivariate prediction formula specifically constructed for an indicated time interval in order to obtain a prediction value (L), where a L value less than a threshold L* value indicates that menarche will not occur within the time interval and a L greater than the threshold L* value indicates that menarche will occur within the specified time interval.
  • a method of predicting that menarche is not anticipated within a known time interval in a human female comprises a) measuring the level of at least one hormone in a saliva sample from said female; and b) comparing the level of at least one hormone to a selected H* value to determine if menarche is not anticipated within said known time interval.
  • the hormone to be measured in step a) is selected from the group consisting of testosterone, 17-beta estradiol, progesterone, 17- ⁇ hydroxyprogesterone, and combinations thereof.
  • the female is in Tanner Stage HI-IV.
  • the hormone to be measured is testosterone and the selected H* value for testosterone is in the range between between 0.01 and 0.05 nanograms per milliliter (ng/ml). If the measured value of testosterone does not exceed said selected H* value for testosterone then menarche is not anticipated within said known time interval.
  • the hormone to be measured is 17-beta estradiol and the selected
  • H* value for 17-beta estradiol is in the range between 0.5 and 10.0 picograms per milliliter
  • the hormone to be measured is progesterone and the selected H* value for progesterone is in the range between 0.01 and 0.05 ng/ml. If the measured value of progesterone does not exceed said selected H* value for progesterone then menarche is not anticipated within said known time interval.
  • the hormone is 17-hydroxyprogesterone and the selected H* value for 17-hydroxyprogesterone is in the range between 0.01 and 0.10 ng/ml. If the measured value of 17-hydroxyprogesterone does not exceed said selected H* value for 17- hydroxyprogesterone then menarche is not anticipated within said known time interval.
  • the saliva sample is collected by the female.
  • the female measures the level of at least one hormone in a saliva sample and compares the level of at least one hormone to a selected H* value to determine if menarche is not anticipated within said known time interval in her own home.
  • the steps of a) measuring the level of at least one hormone in a saliva sample from said female; and b) comparing the level of at least one hormone to a selected H* value to determine if menarche is not anticipated within said known time interval are completed with the use of an immunoassay.
  • a visual color change of the immunoassay indicates a positive or negative result.
  • the known time interval is between 1 to 60 days, or between 1 to
  • the saliva sample is obtained by spitting saliva into a tube, or by swabbing saliva from the mouth, or by depositing saliva onto a test strip.
  • a method of predicting that menarche is anticipated within a known time interval in a human female comprises a) measuring the level of at least one hormone in a saliva sample from said female; and b) comparing the level of at least one hormone to a selected H* value to determine if menarche is anticipated within said known time interval.
  • the hormone to be measured in step a) is selected from the group consisting of testosterone, 17-beta estradiol, progesterone, 17- ⁇ hydroxyprogesterone, and combinations thereof.
  • the female is in Tanner Stage III-IV.
  • the hormone to be measured is testosterone and the selected H* value for testosterone is in the range between 0.01 and 0.05 ng/ml. If the measured value of testosterone exceeds said selected H* value for testosterone then menarche is anticipated within said known time interval.
  • the hormone to be measured is 17-beta estradiol and the selected
  • H* value for 17-beta estradiol is in the range between between 0.5 and 10.0 pg/ml. If the measured value of 17-beta estradiol exceeds said selected H* value for 17-beta estradiol then menarche is anticipated within said known time interval.
  • the hormone to be measured is progesterone and the selected H* value for progesterone is in the range between 0.01 and 0.05 ng/ml. If the measured value of progesterone exceeds said selected H* value for progesterone then menarche is anticipated within said known time interval.
  • the hormone is 17-hydroxyprogesterone and the selected H* value for 17-hydroxyprogesterone is in the range between 0.01 and 0.10 ng/ml. If the measured value of 17-hydroxyprogesterone exceeds said selected H* value for 17- hydroxyprogesterone then menarche is anticipated within said known time interval.
  • the saliva sample is collected by the female.
  • the female measures the level of at least one hormone in a saliva sample and compares the level of at least one hormone to a selected H* value to determine if menarche is anticipated within said known time interval in her own home.
  • the steps of a) measuring the level of at least one hormone in a saliva sample from said female; and b) comparing the level of at least one hormone to a selected H* value to determine if menarche is anticipated within said known time interval are completed with the use of an immunoassay.
  • a visual color change of the immunoassay indicates a positive or negative result.
  • the known time interval is between 1 to 60 days, or between 1 to
  • the saliva sample is obtained by spitting saliva into a tube, or by swabbing saliva from the mouth, or by depositing saliva onto a test strip.
  • a method for predicting that the onset of menarche will not occur in a female within a selected time interval comprises a) determining the concentration of at least one hormone selected from the group consisting of 17-bestradiol, testosterone, progesterone, and 17 ⁇ hydroxy-progesterone (17-OHP) in a saliva sample from said female; b) determining a Body Mass Index (BMI) value for said female; c) applying the concentration of at least one hormone and the BMI value to a multivariate prediction formula to determine an L value; d) comparing the L value obtained in step (c) to a selected L* value, wherein a L value less than the L* value indicates that menarche is not anticipated within said selected time interval.
  • BMI Body Mass Index
  • the levels of at least two hormones, or at least three hormones or at least four hormones are measured and applied to a multivariate prediction formula for two, three, or four hormones, respectively.
  • the L* is selected in a range from -0.05 to +1.60 If said L value is less than the selected L* value then menarche is not anticipated within said selected time interval.
  • the L* is selected from a range from 0.90 to 1.20 when the selected time interval is between 1 to 60 days.
  • the L* is selected from a range from 1.20 to 1.60 when the selected time interval is between 1 to 90 days.
  • the L* is selected from a range from -0.05 to 0.20 when the selected time interval is between 1 to 30 days.
  • the L* is selected from a range from 0.10 to 0.40 when the selected time interval is between 1 to 45 days.
  • the female is in Tanner Stage III-IV.
  • the selected time interval is between 1 to 60 days, or between 1 to
  • a method for predicting the onset of menarche in a female within a selected time interval comprises the steps of: a) determining the concentration of at least one hormone selected from the group consisting of
  • the levels of at least two hormones, or at least three hormones or at least four hormones are measured and applied to a multivariate prediction formula for two, three, or four hormones, respectively.
  • the L* is selected in a range from -0.05 to +1.60. If said L value is greater than the selected L* value then menarche will occur within the selected time interval.
  • the L* is selected from a range from 0.90 to 1.20 when the selected time interval is between 1 to 60 days.
  • the L* is selected from a range from 1.20 to 1.60 when the selected time interval is between 1 to 90 days.
  • the L* is selected from a range from -0.05 to 0.20 when the selected time interval is between 1 to 30 days.
  • the L* is selected from a range from 0.10 to 0.40 when the selected time interval is between 1 to 45 days.
  • the female is in Tanner Stage M-IV.
  • the selected time interval is between 1 to 60 days, or between 1 to
  • kits for predicting that menarche is anticipated in a human female within a known time interval comprises an indicator responsive to a hormonal level of a hormone in a body fluid, wherein the hormone is selected from the group consisting of testosterone, 17-beta estradiol, progesterone, and 17- hydroxyprogesterone.
  • the indicator provides a positive test result when any one of the following conditions are met: a hormonal level of testosterone exceeds a value selected in a range between about 0.01 and 0.05 ng/ml; a hormonal level of 17-beta estradiol exceeds a value selected in a range between about 0.5 and 10.0 pg/ ml; a hormonal level of progesterone exceeds a value selected in a range between about 0.01 and 0.05 ng/ml; a hormonal level of 17-hydroxyprogesterone exceeds a value selected in a range between about
  • the kit is provided for the known time interval between 1 to 60 days, or between 1 to 90 days, or between 1 to 45 days, or between 1 to 30 days.
  • the kit is provided for a female in Tanner stage III or IV, and may, for example, comprise a chart specifying how to determine Tanner Stage
  • kits for predicting that menarche is not anticipated in a human female within a known time interval comprises an indicator responsive to a hormonal level of a hormone in a body fluid, wherein said hormone is selected from the group consisting of testosterone, 17-beta estradiol, progesterone, and 17- hydroxyprogesterone.
  • the indicator provides a positive test result when any one of the following conditions are met: a hormonal level of testosterone does not exceed a value selected in a range between about 0.01 and 0.05 ng/ml; a hormonal level of 17-beta estradiol does not exceed a value selected in a range between about 0.5 and 10.0 pg/ml; a hormonal level of progesterone does not exceed a value selected in a range between about 0.01 and 0.05 ng/ml; a hormonal level of 17-hydroxyprogesterone does not exceed a value selected in a range between about 0.01 and 0.10 ng/ml.
  • the kit is provided for the known time interval between 1 to 60 days, or between 1 to 90 days, or between 1 to 45 days, or between 1 to 30 days.
  • the kit is provided for a female in Tanner stage III or IV, and may, for example, comprise a chart specifying how to determine Tanner Stage.
  • Figures IA and IB show example logistic regression formulas and calculated threshold cutoff values, H* and L*, for a 60 day prediction of menarche.
  • Figure IA, column 1 is labeled "predictors" and indicates either no predictors or the predictors of Body Mass Index (BMI), 17- ⁇ estradiol (E2), testosterone (TEST), progesterone (PROG), 17 hydroxy- progesterone (OHP) and combinations thereof.
  • BMI Body Mass Index
  • E2 17- ⁇ estradiol
  • TEST testosterone
  • PROG progesterone
  • OHP 17 hydroxy- progesterone
  • Figure IA column 2, labeled receiver operator characteristic (ROC) Area shows the area under the ROC curve for the univariate and multivariate predictor logistic regression formulas, with its standard error of estimate to the right (column 3, SE, a "plus-or-minus" index indicating the precision attached the reported value of ROC area). Threshold values are displayed in the next two columns: column 4, H* values labeled "1-vbl cutoff; and column 5, L* values labeled "formula cutoff.” Columns 6, 8 and 10 show the false positive rate (FPR), the positive predictive value (PPV), and the negative predictive value (NPV), respectively.
  • FPR false positive rate
  • PPV positive predictive value
  • NPV negative predictive value
  • Figure IB Logistic regression formulas constructed for the various predictors are shown in Figure IB.
  • Figures 2A and 2B show example logistic formulas and calculated threshold cutoff values, H* and L*, for a 30 day prediction of menarche. The figures are laid out as described in the legend for figure IA and IB.
  • Figure 3A and 3B show example logistic formulas and calculated threshold cutoff values, H* and L*, for a 45 day prediction of menarche. The figures are laid out as described in the legend for figure IA and IB.
  • Figure 4 A and 4B show example logistic formulas and calculated threshold cutoff values, H* and L*, for a 90 day prediction of menarche. The figures are laid out as described in the legend for figure IA and IB.
  • Figure 5 shows a graphical representation of the negative predictive values (NPV) for the time interval of 1 to 60 days for various univariate and multivariate prediction models described in Example 1.
  • the negative predictive values, depicted on the Y-axis, indicate the probability that a girl who is predicted not to reach menarche within 60 days will not do so.
  • the negative predictive values for each model depicted, with the exception of BMI is above 90%.
  • Figure 6 shows a graphical representation of the positive predictive values (PPV) for the time interval of 1 to 60 days for various univariate and multivariate prediction models described in Example 1.
  • the positive predictive values, depicted on the Y-axis, indicate the probability that a girl who is predicted to reach menarche within 60 days will actually do so.
  • the positive predictive values for each model depicted, with the exception of BMI is about 50%.
  • Figure 7 shows a graphical representation of the negative predictive values (NPV) for the time interval of 1 to 30 days for various univariate and multivariate prediction models described in Example 1.
  • the negative predictive values, depicted on the Y-axis, indicate the probability that a girl who is predicted not to reach menarche within 30 days will not do so.
  • Figure 8 shows a graphical representation of the positive predictive values (PPV) for the time interval of 1 to 30 days for various univariate and multivariate prediction models described in Example 1.
  • the positive predictive values depicted on the Y-axis, indicate the probability that a girl who is predicted to reach menarche within 30 days will actually do so.
  • Figure 9 shows a graphical representation of the negative predictive values (NPV) for the time interval of 1 to 45 days for various univariate and multivariate prediction models described in Example 1.
  • the negative predictive values, depicted on the Y-axis indicate the probability that a girl who is predicted not to reach menarche within 45 days will not do so.
  • the negative predictive values for each model depicted, with the exception of BMI alone is above 90%.
  • Figure 10 shows a graphical representation of the positive predictive values (PPV) for the time interval of 1 to 45 days for various univariate and multivariate prediction models described in Example 1.
  • the positive predictive values, depicted on the Y-axis, indicate the probability that a girl who is predicted to reach menarche within 45 days will actually do so.
  • Figure 11 shows a graphical representation of the negative predictive values (NPV) for the time interval of 1 to 90 days for various univariate and multivariate prediction models described in Example 1.
  • the negative predictive values depicted on the Y-axis, indicate the probability that a girl who is predicted not to reach menarche within 90 days will not do so.
  • the univariate and multivariate predictors on the X-axis Using the cut off values described in Figure 1 , the negative predictive values for the models ranges from about 60%- 90%.
  • Figure 12 shows a graphical representation of the positive predictive values (PPV) for the time interval of 1 to 90 days for various univariate and multivariate prediction models described in Example 1.
  • the positive predictive values, depicted on the Y-axis indicate the probability that a girl who is predicted to reach menarche within 90 days will actually do so.
  • the univariate and multivariate predictors on the X-axis The univariate and multivariate predictors on the X-axis.
  • Figure 13 shows a ROC curved fitted for the multivariate logistic regression formula for the selected period of 1 to 60 days before the onset of menarche and the predictor variables are 17-beta estradiol (E2), testosterone (TEST) and body mass index (BMI).
  • E2 17-beta estradiol
  • TEST testosterone
  • BMI body mass index
  • Figure 14 shows the graphical experimental data of L values calculated using the multivariate logistic regression formulae for the predictor variables 17-beta estradiol (E2), testosterone (TEST) and body mass index (BMI).
  • the X-axis represents the period of 1 to 150 days before the onset of menarche and the time of onset of menarche is set at 0 time.
  • the Y-axis represents the L values.
  • the L* value of 1.025 is selected from Figure 13's ROC curve where the sensitivity is 80% and is specificity is also 80%.
  • Figure 15 shows a schematic diagram of a method of the invention for determining that the level of a hormone in saliva is greater than a predetermined threshold level using a simple one-step analytical strategy.
  • Figure 16 shows a schematic diagram showing the interpretation of the results obtained using the simple one-step analytical strategy shown in figure 15.
  • Figure 17 shows a schematic diagram showing a simple one-step analytical strategy where the levels of four hormones are determined simultaneously using four different test strips.
  • Figure 18 shows a schematic diagram showing a simple one-step analytical strategy where the levels of three hormones are determined simultaneously on the same membrane and test strip.
  • Figure 19 shows a schematic diagram showing an alternate version of the method of the invention using a simple one-step analytical strategy.
  • Figure 20 shows a schematic diagram showing the interpretation of the results obtained using the simple one-step analytical strategy shown in figure 19.
  • Figure 21 shows a schematic diagram showing an alternate simple one-step analytical strategy where the levels of four hormones are determined simultaneously using four different test strips.
  • Figure 22 shows a schematic diagram showing an alternate simple one-step analytical strategy where the levels of three hormones can be determined simultaneously on the same membrane in a single dipstick test strip.
  • Figure 23 shows a ROC curved fitted for the univariate logistic regression formula for the selected period of 1 to 60 days before the onset of menarche and the predictor variable is
  • Figure 24 shows the graphical experimental data of H values calculated using the univariate logistic regression formulae for the predictor variables 17-beta estradiol.
  • the X- axis represents the period of 1 to 150 days before the onset of menarche and the time of onset of menarche is set at 0 time.
  • the Y-axis represents the H values.
  • the H* value of 8.52 pg/ml is selected from Figure 23 's ROC curve where the sensitivity is 75% and is specificity is also 75%.
  • Embodiments of the invention provide methods and diagnostic kits for reliably predicting that the onset of menarche will or will not, occur in girls and young women within a known time interval, e.g. 1 to 30 days, 1 to 45 days, 1 to 60 days, or 1 to 90 days.
  • a known time interval e.g. 1 to 30 days, 1 to 45 days, 1 to 60 days, or 1 to 90 days.
  • menarche refers to a females first menstrual period.
  • useful prediction formulas can be constructed for specified time intervals, including but not limited to, 1 to 30 days, 1 to 45 days, 1 to 60 days and 1 to 90 days, by applying both univariate and multiple logistic regression analysis to data sets reflecting the levels of salivary hormones present in females before, and at the onset, of menarche.
  • predetermined threshold levels of hormones, H* are determined using univariate logistic regression analysis and these H* levels are used as the threshold level of hormone which serves as a predictor as to whether or not menarche will occur in a female (e.g. a female in Tanner Stage I- V) within a known time interval including, but not limited to, 1 to 30 days, 1 to 45 days, 1 to 60 days, and 1 to 90 days. If a girl's salivary hormone level is below the threshold level, H*, it is anticipated that menarche will not occur within the interval. Alternatively, if a if a girl's salivary hormone level is above a threshold level, H*, it is anticipated that menarche will occur within the selected time interval.
  • a predetermined threshold amount of hormone refers to the amount of hormone determined to be a threshold level of hormone for a female where salivary hormone levels below a threshold amount, H*, indicate that menarche in said female will not occur and salivary hormone levels above a H* indicate that menarche will occur within a selected time interval.
  • a predetermined threshold amount of hormone is determined for a desired interval and a desired outcome by applying univatriate logistic regression analysis to a data set of premenarchal hormone levels determined for each hormone in the selected "known time interval.”
  • the predetermined threshold hormone level is selected to be high or low enough to guarantee a desired sensitivity, e.g. a sensitivity of 80% or greater.
  • H* can be selected with the aid of a receiver operating characteristic curve (a ROC curve) analysis which plots the true positive rate (sensitivity) versus the false positive rate (1 -specificity) ( Figure 23).
  • H* can be selected to render varied probabilities of accuracy for the selected outcome (whether menarche will, or will not occur) , e.g. 45-50%, 50-55%, 55-60%, 60-65%, 65-70%, 70-75%, 75-80%, 80-90%, 90-95%, or 95-100%.
  • the H* value of 8.52 pg/ml ( Figure 24) is selected from the ROC curve ( Figure 23) for the fitted univariate logistic regression formula where 17-beta estradiol is the sole predictor variable and a specified time interval of 1 to 60 days such that the sensitivity is about 75% and is specificity is also 75%.
  • predetermined threshold amounts of hormone, H* were selected using logistic regression analysis of weekly salivary hormonal levels in a sample set of 63 Tanner Stage III-IV females for the intervals of 1 to 30 days, 1 to 45 days, 1 to 60 days, and 1 to 90 days (see Figures 1-4, and Example 1).
  • Figure IA shows the predetermined threshold hormone levels, H*, selected for 17— ⁇ estradiol (E2, pg/ml), testosterone (TEST, ng/ml), progesterone (PROG, ng/ml), and 17 ⁇ hydroxy-progesterone (OHP, ng/ml) such that levels below the threshold indicate that there is about 93-94% probability (NPV values from about 0.93 to about 0.94, column 10) that the Tanner Stage III-IV female will not reach menarche within 60 days.
  • H* predetermined threshold hormone levels
  • H* threshold levels were about 8.5 g/ml for 17— ⁇ estradiol, about 12.4 pg/ml for testosterone, about 36.0 pg/ml for progesterone, and about 76.6 pg/ml for 17 ⁇ hydroxy-progesterone (17-OHP).
  • Threshold levels were also determined for these hormones using the selected time intervals of 1 to 30 days (Fig. 2A), 1 to 45 days (Fig. 3A), and 1 to 90 days (Fig 4A) and these levels were correlated to the probabilities that menarche will (PPV) or will not occur (NPV) within the selected time interval (see Figures 1 A-4A, columns 8 and 10).
  • a method of predicting that menarche is not anticipated within a specified time interval in a human female involves measuring the level of at least one hormone in a saliva sample from a female and comparing the measured level to a selected H* value to determine if menarche is not anticipated within the time interval. A salivary hormone level below H* indicates menarche will not occur within the specified time interval.
  • ranges of H* values for the hormones testosterone, 17-beta estradiol, progesterone, and 17- ⁇ hydroxyprogesterone for the specified time interval are provided.
  • the H* value for testosterone can be selected from a range between 0.01 and 0.05 ng/ml and the H* value of 17-beta estradiol is selected from a range between 0.5 and 10.0 pg/ml.
  • the H* values for progesterone is selected from a range between 0.01 and 0.05 ng/ml and the H* value for 17-hydroxyprogesterone is be selected from a range between 0.01 and 0.10 ng/ml.
  • a method of predicting that menarche is not anticipated within 1 to 60 days in a human female in Tanner Stage III-IV involves measuring the level of at least one hormone (e.g. 17- ⁇ estradiol, testosterone, progesterone, and/or 17 q hydroxy-progesterone) in a saliva sample from the female and comparing the measured level to a selected H* value to determine if menarche is not anticipated within the time interval. A level of hormone below the selected H* value indicates that menarche will not occur with in 60 days.
  • at least one hormone e.g. 17- ⁇ estradiol, testosterone, progesterone, and/or 17 q hydroxy-progesterone
  • the selected H* threshold amount of hormone can range from 5.00 pg/ml -15.00 pg/ml for 17-beta-estradiol, from 8.00 pg/ml-18 pg/ml for testosterone, from 30 pg/ml-40 pg/ml for progesterone, and from 70 pg/ml-80 pg/ml for 17 hydroxy-progesterone (17-OHP).
  • H* is about 8.5 pg/ml 17 beta- estradiol, about 0.012 ng/ml testosterone, about 0.036 ng/ml progesterone, and about 0.076 ng/ml 17 hydroxy-progesterone (17-OHP).
  • a method of predicting that menarche is anticipated in a human female within a known time interval comprises measuring the level of at least one hormone in a saliva sample from a female and comparing the level hormone to a selected H* value to determine if menarche is anticipated within said known time interval. A salivary hormone level above the selected H* indicates that menarche will occur within the specified time interval.
  • the salivary hormone to be measured hormone is selected from the group consisting of testosterone, 17-beta estradiol, progesterone, 17- ⁇ hydroxyprogesterone, and combinations thereof.
  • ranges of H* values for the hormones testosterone, 17-beta estradiol, progesterone, and 17- ⁇ hydroxyprogesterone for the specified time interval are provided.
  • the H* value for testosterone is selected from a range between 0.01 and 0.05 ng/ml and the H* value of 17-beta estradiol is selected from a range between 0.5 and 10.0 pg/ml.
  • the H* values for progesterone is selected from a range between 0.01 and 0.05 ng/ml and the H* value for 17-hydroxyprogesterone is selected from a range between 0.01 and 0.10 ng/ml.
  • the female to be tested for the onset of menarche is in Tanner Stage III or IV.
  • Tanner Stage III the terms “Tanner Stage I”, “Tanner Stage II”, “Tanner Stage III”, “Tanner Stage IV” and “Tanner Stage V” refer to the female Tanner stages of breast development and pubic hair development.
  • Tanner stages of breast development include Tanner Stage I, a preadolescent, the nipple elevates; Tanner Stage II, the breast bud develops and the breast tissue and nipple arise as a single mound of tissue; Tanner Stage III, the single mound enlarges; Tanner Stage IV, a secondary mound develops, with the nipple and areola projecting above the breast tissue; Tanner Stage V, the areola regresses to form a smooth contour with the rest of the breast tissue.
  • Tanner stages of pubic hair include Tanner Stage I, no pubic hair; Tanner Stage II, a small amount of long, downy hair with slight pigmentation on the labia majora; Tanner Stage III, hair becomes more coarse and curly, and begins to extend laterally; Tanner Stage IV, adult-like hair quality, extending across pubis but sparing medial thighs; and Tanner Stage V, hair extends to medial surface of the thighs.
  • Saliva samples can be collected by any means known to those skilled in the art, e.g. by spitting saliva into a tube, by swabbing saliva from the mouth, or by depositing saliva onto a test strip.
  • any hormone present in saliva that is useful to predict menarche in a female can be measured and used to predict menarche using methods of the invention.
  • Hormonal levels in the saliva sample can be measured by any means known to those skilled in the art.
  • the levels of salivary hormone/s is/are measured using an immunoassay.
  • Such an immunoassay can provide a positive or negative result as an indication as to whether or not menarche will occur.
  • Means for measuring the levels of salivary hormones are described in more detail herein under the subheading Measuring levels of Salivary Hormone.
  • a female can collect her own saliva sample and measure the levels of hormones in her saliva in the privacy of her own home. For example using a home test kit.
  • multivariate prediction formulas can been constructed for varied time intervals using multiple logistic regression analysis with incidence of menarche as the dichotomous dependent variable and combinations of hormone levels and Body Mass Index as independent variables. Such multivariate prediction formulas can reliably determine whether or not menarche will occur in a female within a specified time interval.
  • BMI Body Mass Index
  • the L* value is selected to provide the desired sensitivity and specificity. It can be selected from a receiver operator characteristic (ROC) curve that has a c-statistic or area under the ROC curve of at least 0.85 which is a measure of the predictive strength of a multi- variant logistic regression, the prediction formula.
  • ROC receiver operator characteristic
  • a ROC curve of a multi-variant logistic regression that gives a c-statistic ranging from about 0.85 to 0.99 is indication of the strong predictive strength of that multi-variant logistic regression.
  • the L* value is selected from a range on the ROC curve that provides the highest degree of sensitivity while keeping the false positive rate to a minimum.
  • the sensitivity is the probability that a girl who is going to reach menarche will be correctly predicted to do so and the specificity is the probability that a girl who is not going to reach menarche will be correctly predicted not to do so.
  • a range of sensitivity may be around 75-95% with a specificity of 75-95%.
  • a prediction formula for three variables: 17 ⁇ -estradiol, testosterone, and BMI, and for menarche onset between 1-60 days has a good c-statistic or area under the ROC curve of 0.90 (Fig. 13).
  • variable combinations BMI andl7- ⁇ estradiol (E2); BMI and testosterone (TEST); BMI and progesterone (PROG); BMI and 17 hydroxy-progesterone (OHP); 17- ⁇ estradiol (E2) and testosterone; 17- ⁇ estradiol (E2) and progesterone (PROG) ; 17- ⁇ estradiol (E2) and 17 hydroxy-progesterone (OHP); progesterone and testosterone; progesterone and 17 hydroxy-progesterone ; 17 hydroxy-progesterone (17-OHP) and testosterone; 17- ⁇ estradio
  • Multivariate prediction formulas and L* threshold values for the prediction of menarche within the intervals of 1 to 30 days, 1 to 45 days, 1 to 60 days and 1 to 90 days for the above combinations are indicated in Figures 1-4.
  • L* is indicated by the formula cut off values listed in column 5 of Figures IA, 2 A, 3 A and 4 A.
  • the multivariate logistic formulas to obtain L values for each selected time interval are listed in Figures IB, 2B, 3B, and 4B.
  • a method for predicting that the onset of menarche will not occur within a selected time interval in a female subject is provided.
  • the method comprises a) determining the level of at least one hormone selected from the group consisting of 17- ⁇ estradiol, testosterone, progesterone, and ⁇ 1 a hydroxy-progesterone (17- OHP) in a saliva sample from said female subject; b) determining a Body Mass Index (BMI) value for said female; c) applying the level of at least one hormone and the BMI value to a multivariate prediction formula to determine an L value; and d) comparing the L value obtained in step (c) to a selected L* level, where a L value less than L* indicates that menarche will not occur in the selected time interval in said female.
  • the selected time interval is 1 to 30 days.
  • the selected time interval is 1 to 45 days.
  • the selected time interval is 1 to 60 days.
  • the selected time interval is 1 to 90 days.
  • the female is in Tanner stage III or IV.
  • the L* values is selected from a range between -0.05 to
  • the L* is selected from a range from 0.90 to 1.20 when the selected time interval is between 1 to 60 days. In another preferred embodiment, the L* is selected from a range from 1.20 to 1.60 when the selected time interval is between 1 to 90 days. In yet another preferred embodiment, the L* is selected from a range from -0.05 to 0.20 when the selected time interval is between 1 to 30 days. In another preferred embodiment, the L* is selected from a range from 0.10 to 0.40 when the selected time interval is between 1 to 45 days. [00102] In one embodiment, a method for predicting that the onset of menarche will occur within a selected time interval in a female subject is provided.
  • the method comprises a) determining the level of at least one hormone selected from the group consisting of 17- ⁇ estradiol, testosterone, progesterone, and 17 ⁇ hydroxy-progesterone (17-OHP) in a saliva sample from said female subject; b) determining a Body Mass Index (BMI) value for said female; c) applying the level of at least one hormone and the BMI value to a multivariate prediction formula to determine an L value; and d) comparing the L value obtained in step (c) to a selected L* level, where a L value greater than L* indicates that menarche will occur in the selected time interval in said female.
  • the selected time interval is 1 to 30 days.
  • the selected time interval is 1 to 45 days.
  • the selected time interval is 1 to 60 days.
  • the selected time interval is 1 to 90 days.
  • the female is in Tanner stage III or IV.
  • the L* values is selected from a range between -0.05 to
  • the L* is selected from a range from 0.90 to 1.20 when the selected time interval is between 1 to 60 days. In another preferred embodiment, the L* is selected from a range from 1.20 to 1.60 when the selected time interval is between 1 to 90 days. In yet another preferred embodiment, the L* is selected from a range from -0.05 to 0.20 when the selected time interval is between 1 to 30 days. In another preferred embodiment, the L* is selected from a range from 0.10 to 0.40 when the selected time interval is between 1 to 45 days. [00104] Measuring levels of Salivary Hormone
  • the level of hormone in saliva is measured to obtain a determination of whether or not menarche will occur.
  • the salivary hormone levels can be measured using any assay known to those skilled in the art, including, but not limited to, Enzyme-Linked Immunosorbent Assay (ELISA), immunoprecipitation assays, radioimmunoassay, mass spectrometry, Western Blotting, and via dipsticks using conventional technology.
  • the level of hormone in the saliva should be measured in the same manner as the predetermined threshold level is measured.
  • the levels of hormone can be represented in arbitrary units dependent upon the assay used to measure the levels of hormone, e.g., the intensity of the signal from the detectable label can correspond to the amount of hormone present (e.g. as determined by eye, densitometry, an ELISA plate reader, a luminometer, or a scintillation counter).
  • the levels of hormone present in saliva samples can be determined using a ligand that specifically binds to the hormone, e.g, a synthetic peptide, chemical, or antibody.
  • the ligand is preferably detectably labeled.
  • immunoassays using antibodies are used to measure the levels of hormone in saliva.
  • the term "antibody” is intended to include immunoglobulin molecules and immunologically active determinants of immunoglobulin molecules, e.g., molecules that contain an antigen binding site which specifically binds (immunoreacts with) to the hormone to be measured.
  • the term “antibody” is intended to include whole antibodies, e.g., of any isotype (IgG, IgA, IgM, IgE, etc), and includes fragments thereof which are also specifically reactive with the hormone to be measured, e.g.
  • Antibodies can be fragmented using conventional techniques.
  • the term "antibody” includes segments of proteolytically-cleaved or recombinantly -prepared portions of an antibody molecule that are capable of selectively reacting with a certain protein.
  • proteolytic and/or recombinant fragments include Fab, F(ab')2, Fab' , Fv, dAbs and single chain antibodies (scFv) containing a VL and VH domain joined by a peptide linker.
  • the scFv's may be covalently or non-covalently linked to form antibodies having two or more binding sites.
  • antibody includes polyclonal, monoclonal, or other purified preparations of antibodies and recombinant antibodies.
  • the term “antibody” is further intended to include humanized antibodies, bispecific antibodies, and chimeric molecules having at least one antigen binding determinant derived from an antibody molecule. In one embodiment, the antibody is detectably labeled.
  • Antibodies to the hormones can be generated using methods known to those skilled in the art. Alternatively, commercially available antibodies can be used. Antibodies to 17- ⁇ estradiol, testosterone, progesterone, and/or 17 hydroxy-progesterone (17-OHP) are commercially available.
  • detectably labeled includes antibodies that are labeled by a measurable means and include, but are not limited to, antibodies that are enzymatically, radioactively, fluorescently, and chemiluminescently labeled. Antibodies can also be labeled with a detectable tag, such as c-Myc, HA, VSV-G, HSV, FLAG, V5, HIS, or biotin. [00111] In the diagnostic methods of the invention that use an antibody for the detection of hormone levels, the level of hormone present in the saliva samples correlates to the intensity of the signal emitted from the detectably labeled antibody. [00112] In one embodiment, the antibody is detectably labeled by linking the antibody to an enzyme.
  • Enzymes which can be used to detectably label the antibodies of the present invention include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-V-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta- galactosidase, ribonuclease, urease, catalase, glucose-VI-phosphate dehydrogenase, glucoamylase and acetylcholinesterase. Chemiluminescence is another method that can be used to detect an antibody.
  • Detection may also be accomplished using any of a variety of other immunoassays.
  • radioactively labeling an antibody it is possible to detect the antibody through the use of radioimmune assays.
  • the radioactive isotope can be detected by such means as the use of a gamma counter or a scintillation counter or by audoradiography.
  • Isotopes which are particularly useful for the purpose of the present invention are H, I, 35 S, 14 C, and preferably 125 I.
  • fluorescent labeling compounds include CYE dyes, fluorescein isothiocyanate, rhodamine, phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.
  • An antibody can also be detectably labeled using fluorescence emitting metals such as 15 Eu, or others of the lanthanide series. These metals can be attached to the antibody using such metal chelating groups as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA).
  • DTPA diethylenetriaminepentaacetic acid
  • EDTA ethylenediaminetetraacetic acid
  • An antibody also can be detectably labeled by coupling it to a chemiluminescent compound. The presence of the chemiluminescent-antibody is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
  • chemiluminescent labeling compounds are luminol, luciferin, isoluminol, theromatic acridinium ester, imidazole, acridinium salt and oxalate ester.
  • the levels of hormone in saliva are detected by ELISA assay.
  • ELISA assay There are different forms of ELISA which are well known to those skilled in the art, e.g. standard ELISA, competitive ELISA, and sandwich ELISA. The standard techniques for ELISA are described in "Methods in Immunodiagnosis", 2nd Edition, Rose and Bigazzi, eds. John Wiley & Sons, 1980; Campbell et al., "Methods and Immunology", W. A. Benjamin, Inc., 1964; and Oellerich, M. 1984, J. Clin. Chem. Clin. Biochem., 22:895-904.
  • the levels of salivary hormone are determined by contacting a saliva sample with a first antibody that specifically binds to a hormone to be measured under conditions permitting formation of a complex between the antibody and said hormone (e.g. 17- ⁇ estradiol, testosterone, progesterone, and 17 ⁇ hydroxy-progesterone (17- OHP)).
  • a complex between the antibody and said hormone e.g. 17- ⁇ estradiol, testosterone, progesterone, and 17 ⁇ hydroxy-progesterone (17- OHP)
  • the amount of complex formed is then measured as a measure of the level of the hormone, and the amount of complex formed is compared to the amount of complex formed between the first antibody and a predetermined threshold amount of the hormone.
  • a level below the threshold amount of hormone indicates that menarche will not occur, while a level above the threshold amount of hormone indicates that menarche will occur.
  • the first antibody is detectably labeled. Detectably labeling the first antibody is appropriate for use, for example, in standard ELISA assays where hormone is absorbed to an ELISA plate, or in Western Blot analysis, or certain dipstick analyses.
  • the first antibody is immobilized on a solid support, for example, when using a "Sandwich Elisa" or a dipstick analysis, then the amount of complex formed can measured by detecting binding of a second antibody that specifically binds to the hormone (e.g. 17- ⁇ estradiol, testosterone, progesterone, and 1 /hydroxy-progesterone (17- OHP)) under conditions permitting formation of a complex between the second antibody and the hormone, wherein the second antibody does not substantially cross-react with the first antibody, and wherein the second antibody is detectably labeled.
  • the hormone e.g. 17- ⁇ estradiol, testosterone, progesterone, and 1 /hydroxy-progesterone (17- OHP
  • Any solid support can be used, including but not limited to, nitrocellulose, solid organic polymers, such as polystyrene, or laminated dipstcks such as described in U.S. patent 5,550,375.
  • the levels of two hormones defining a first and a second hormone are measured using at least two antibodies specific to each hormone to be measured. Each antibody specifically reacts either the first hormone or the second hormone to be measured while not substantially cross-reacting with the other hormones to be measured.
  • the levels of three hormones defining a first hormone, a second hormone, and a third hormone are measured using at least three antibodies specific to each hormone to be measured, wherein each antibody specifically reacts either the first hormone, the second hormone, or the third hormone to be measured while not substantially cross-reacting with the other hormones to be measured.
  • the levels of four hormones defining a first, a second, a third hormone and a fourth hormone are measured using at least four antibodies specific to each hormone to be measured, wherein each antibody specifically reacts either the first hormone, the second hormone, the third hormone, or the fourth hormone to be measured while not substantially cross-reacting with the other hormones to be measured.
  • the hormones are selected from the group consisting of
  • Embodiments of the invention further provide for diagnostic kits and products of manufacture comprising the diagnostic kits.
  • the kits can comprise a means for predicting that menarche is anticipated in a human female, they can comprise a means for predicting that menarche is not anticipated in a human female within a known time interval, or they can comprise both.
  • the known time interval can be, for example, between 1 to 60 days, or between 1 to 90 days, or between 1 to 30 days, or between 1 to 45 days. Other known time intervals can be used.
  • the kit comprises an indicator responsive to a hormonal level of a hormone in a body fluid, wherein the hormone is selected from the group consisting of testosterone, 17-beta estradiol, progesterone, and 17-hydroxyprogesterone.
  • the indicator provides a positive test result when any one of the following conditions are met: a hormonal level of testosterone exceeds a value selected in a range between about 0.01 and 0.05 ng/ml; a hormonal level of 17-beta estradiol exceeds a value selected in a range between about 0.5 and 10.0 pg/ml; a hormonal level of progesterone exceeds a value selected in a range between about 0.01 and 0.05 ng/ml; a hormonal level of 17-hydroxyprogesterone exceeds a value selected in a range between about 0.01 and 0.10 ng/ml.
  • the kit comprises an indicator responsive to a hormonal level of a hormone in a body fluid, wherein said hormone is selected from the group consisting of testosterone, 17-beta estradiol, progesterone, and 17-hydroxyprogesterone.
  • the indicator provides a positive test result when any one of the following conditions are met: a hormonal level of testosterone does not exceed a value selected in a range between about 0.01 and 0.05 ng/ml; a hormonal level of 17-beta estradiol does not exceed a value selected in a range between about 0.5 and 10 pg/ml; a hormonal level of progesterone does not exceed a value selected in a range between about 0.01 and 0.05 ng/ml; a hormonal level of 17- hydroxyprogesterone does not exceed a value selected in a range between about 0.01 and 0.10 ng/ml.
  • the body fluid can be saliva, tears, urine, sweat, blood plasma, or vaginal secretion.
  • the kits can further comprise cups or tubes, or any other collection device for sample collection of the body fluids.
  • kits further comprise charts, for example describing the Five Tanner Stages of development, describing the interpretation of test results, or describing how to calculate body mass index.
  • An article of manufacture comprising a package of tampons or menstrual pads and a kit comprising a means for predicting the onset of menarche for a female subject is also contemplated.
  • Example I Levels of salivary 17- ⁇ estradiol, testosterone, progesterone, and
  • 17-hydroxyprogesterone (17-OHP) and BMI alone or in combination, can predict menarche in healthy premenarcheal girls.
  • the fitted logistic model takes the following form in the case of a single hormone:
  • the rule is slightly more complex: a) Ssecify a threshold logit value b) If a girl's hormone levels and/or BMI, entered into the logistic formula, produce a value above the threshold, predict menarche within the interval; if below, predict no menarche.
  • the strength of prediction can be summarized by several different statistical parameters, all derived from the fitted model. The most general is the area under the receiver operating characteristic (ROC) curve, an index between 0.5 and 1. A value of 0.5 is no better than random chance; a value of 1 is perfect prediction.
  • ROC receiver operating characteristic
  • FPR false positive rate
  • Example II Provided are two examples of how the prediction formulas derived in Example I would be used for individual girls.
  • estradiol (E2) to predict menarche within 60 days.
  • the cutoff value is 8.5 pg/ml (see Figure IA, E2, column H*, and Figure 24).
  • Two girls are determined to be eligible for testing, both being of appropriate age and Tanner stage (Tanner Stage III-IV).
  • Girl #1 shows salivary estradiol of 10.5 pg/ml. Because this value is above the cutoff, we predict she will reach menarche within 60 days. The chance that this will actually come to pass is 44.8% (PPV), see Figure IA, column 8, predictor E2.
  • Girl #2 shows salivary estradiol of 6.1 pg/ml, which is below the cutoff. We therefore predict she will not reach menarche within 60 days. The chance that this prediction will hold is 93.8% (NPV), see Figure IA, column 10, predictor E2.
  • (B) Suppose we want to use 17-hydroxyprogesterone (OHP) and BMI to predict menarche within 60 days.
  • the logistic formula is 0.8111 + 0.469 x log(OHP) + 0.045 x BMI, (see Figure IB, predictors OHP and BMI, row 8, logistic formula) and the cutoff value is 0.9872 (see Figure IA, predictors OHP and BMI, L* value, row 9).
  • Two eligible girls take the test. Girl #1 has salivary OHP 0.057 ng/ml and BMI 19.8 kg/m2.
  • the formula produces a logit value of 1.1186, larger than the threshold, so that we predict menarche within 60 days.
  • a single protein binding membrane strip is divided into three separate regions: a sample (S) position at one end of the membrane, a test (T) position located at the middle of the membrane, and a control (C) position found at the opposite end the membrane ( Figure 15).
  • S sample
  • T test
  • C control
  • Located at S is a defined quantity of dehydrated anti-hormone antibody.
  • the antibody can be conjugated to colloidal gold beads or latex beads for visualization purposes.
  • T there is a defined quantity of hormone immobilized on the membrane.
  • C there is another immobilized protein, an antibody immunoreactive to the anti-hormone antibody located at the S position ( Figure 15).
  • the defined quantity of dehydrated anti-hormone antibody at S position is such that there is just enough antibody to bind the ligand hormone from the saliva when the salivary hormone is at the threshold level. Therefore when the salivary hormone is at or above the threshold H* level, all the anti-hormone antibody at the S position will be bound to the hormone in the form of hormone-antibody conjugates; there will be no free anti -hormone antibody present.
  • the choice of the anti-hormone antibody placed at the S position can be any antibody that is specially immunoreactive to any of the hormones of interest.
  • the antibody can be monoclonal, polyclonal, or a mixture of both monoclonal and polyclonal antibodies.
  • the S position will have only one antibody specially immunoreactive with just that one hormone of interest.
  • the S position will have three different types of anti- hormone antibodies, each type specially immunoreactive to one hormone and does not exhibit cross-reactivity with the other two non-ligand hormones ( Figure 18).
  • the S position is where a sample of saliva is applied. The arrowheads delineate the boundary limit that the sample saliva should not cross on the membrane when applying the saliva to the membrane.
  • the defined quantity of hormone immobilized on the membrane at the T position ( Figure 15) is a quantity that will bind and capture any free anti-hormone antibody from the S position that is not found in a hormone-antibody conjugate form. That quantity of immobilized hormone will serve to immobilize free anti-hormone antibody to the T position during testing. When only one hormone is studied, only one hormone, the same type as the one of interest will be immobilized at the T position. Likewise when three hormones are to be studied simultaneously, all three hormone types will be represented at the T position and at their respective quantities ( Figure 18).
  • the antibody at the C position can be a monoclonal, polyclonal, or a mixture of both monoclonal and polyclonal antibodies.
  • the antibody at the C position can be a monoclonal, polyclonal, or a mixture of both monoclonal and polyclonal antibodies.
  • the salivary hormone of interest When the salivary hormone of interest is at or above the predetermined threshold H* level, the mixture of antibody and hormone will contain all antibody-hormone conjugates and no free anti-hormone antibody.
  • the T position At the T position, there will be no colloidal gold or latex bead labeled anti-hormone antibody, and the area remains clear ( Figure 16, left).
  • the hormone-antibody conjugates At the C position the hormone-antibody conjugates will be bound and captured by the immobilized antibody immunoreactive against the anti-hormone antibody coming from the S position. This will in turn results in a concentration of a colloidal gold or latex bead labeled anti-hormone antibody at the C position and will become visible as colored line at the C position.
  • the C position result serves as a test control that there is functional anti-hormone antibody in the test material and should always be present (Figure 16, right).
  • the one-step analytical strategy of the invention may be designed in a form of a dipstick test strip ( Figure 15B).
  • a dipstick test strip As a dipstick test strip, the strip is dipped into a sample of saliva at the S position end with saliva level not to exceed the boundary limit. The strip is then laid horizontally with the membrane surface facing up on a flat surface. A fixed amount of time is given for the antibody re-hydration, capillary action, and antibody binding reaction to take place.
  • Figure 16 shows a method of using four separate dipstick test strips to test for the four hormones of interest. Each dipstick test strip is labeled to indicate which hormone is being tested.
  • Figure 18 shows an alternate design where three hormones can be studied simultaneously on the same membrane and therefore on the same same dipstick test strip. The positions of the expected results in the T and C positions for each hormone are indicated.
  • FIG. 5 There is the same single protein binding membrane strip that is divided into three separate regions: a sample (S), a test (T), and a control (C) positions, all three spatially arranged as shown in Figure 15 and Figure 19.
  • the S position will contain an excess amount of dehydrate anti -hormone antibody that may be colloidal gold or latex bead labeled.
  • the T position contains a second antibody that is also immunoreactive to the salivary hormone of interest. This second antibody is in excess and is immobilized on the membrane. This second anti -hormone antibody binds a part of the hormone that is different from the part of the hormone that is bound by the first anti-hormone antibody found at the S position.
  • the second antibody at the T position will bind and capture both free unbound hormone and hormone-antibody conjugates, and concentrate them at the T position.
  • the C position contains a defined quantity of hormone immobilized on the membrane. The defined quantity is the predetermined threshold level of the hormone under study.
  • the excess free anti-hormone from the S position arrives and bind the immobilized hormone, gradually becomes concentrated at the C position and will become visible as a colored line at the C position ( Figure 20).
  • An application of a sample of saliva at the S position will re-hydrate the excess amount of anti -hormone antibody there. A fluid mixture of free antibody and hormone-antibody conjugate is formed and will move along the membrane by capillary action towards the T position and then subsequently to the C position.
  • the thickness of the color band at the T and C positions on the same membrane By comparing the thickness of the color band at the T and C positions on the same membrane, one can estimate whether the salivary hormone level is below or greater than the predetermined threshold level of the hormone.
  • the color band at the T position When the salivary hormone level is equal or greater than the threshold level, the color band at the T position will be equal or larger than the color band at the C position respectively ( Figure 20, left).
  • the color band at the T position will be smaller or even absent than the color band at the C position ( Figure 20, middle).
  • the C position band also serves as a test control to confirm that there is functional anti-hormone antibody at the S position and that the functional anti-hormone antibody is derived from the S position ( Figure 20, right).
  • This alternate version of the simple one-step analytical strategy may be designed in the form of a dipstick test strip as shown in Figure 21 where individual strip is used for each hormone and the strips are labeled.
  • Another design is a single test strip where three hormones can be studied simultaneously on the same membrane. The positions of the expected results in the T and C positions for each hormone are indicated.

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Abstract

L'invention concerne des procédés pour prédire le début de la ménarche, à savoir la première menstruation chez la femme, par l'intermédiaire de la mesure des niveaux d'hormones salivaires (par exemple 17-b estradiol, testostérone, progestérone et/ou 17 hydroxy-progestérone (17-OHP)). En particulier, les procédés décrits ici permettent la détermination fiable que la ménarche aura lieu, n'aura pas lieu, dans des intervalles de temps variés, par exemple 1 à 30 jours, 1 à 45 jours, 1 à 60 jours et 1 à 90 jours. Des kits de diagnostic et des produits de fabrication comprenant les kits sont également fournis.
PCT/US2008/002566 2007-02-28 2008-02-27 Procédés de prédiction du début de la ménarche Ceased WO2008106149A1 (fr)

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